Analytical Techniques Utilizing Antibodies: flow cytometry gel electrophoresis immunoprecipitation...

Analytical Techniques Utilizing Antibodies:• flow cytometry• gel electrophoresis• immunoprecipitation (IP)• immunoblotting

• microscopy• immunofluorescence (IFA)• electron microscopy

• ELISA

• antibodies bind proteins with high specificity and affinity• affinity chromatography• analytical techniques

Antibodies

CNBr

Immunoprecipitation• affinity purification based on

isolation of Ag-Ab complexes• analyze by gel electrophoresis• initially based on centrifugation of

large supramolecular complexes• [high] and equal amounts

• isolation of Ag-Ab complexes• fixed S. aureus• protein A-agarose• protein G-agarose

Bacterial proteins that bind IgG (Fc):• protein A (Staphylococcus aureus)• protein G (Streptococcus)• binds more species and subclasses

Typical IP Protocol1. Solubilize antigen• usually non-denaturing• SDS + excess of TX100

2. Mix extract and Ab 3. Add protein G-agarose, etc4. Extensively wash5. Elute with sample buffer6. SDS-PAGE7. Detection• protein stain• radioactivity

Gagarose

Radiolabeling of Proteins

• carried out before IP• metabolic (amino acids or

other precursors + cells)• chemically (eg., iodination)• IP and SDS-PAGE• detect by autoradiography

or fluorography following electrophoresis

• also provides information about synthesis, post-translational events, etc.

Immunoblotting• aka Western Blotting• use Ab to detect protein after

electrophoresis

1. Protein electrophoresis2. Transfer proteins to membrane3. Incubate membrane with antibody4. Extensive washing5. Detect bound antibody

- radioactive 2nd Ab or protein A - ELISA

Enzyme-Linked ImmunoSorbent Assay

Detection by Chemiluminescence

Western Blot vs Immunoprecipitation

• Experimental Design• eg., synthesis (IP)

• Ag concentration• IP better for low

abundance proteins• Ag solubility• Western for insoluble

proteins• Ab recognition• conformational

dependent epitopes• 4o structure

• carry out IP and electrophoresis • transfer to membrane• use same or different Ab

against Ag in blotting• need ‘no extract’ control

Combined Immunoprecipitation and Immunoblotting

• antigen detection, characterization, (quantification?)• antibody detection, characterization, (quantification?)

Immunoassay Uses